From 1981 to date, there have been eight thousand (8,000) people diagnosed as having acquired immune deficiency syndrome (AIDS). N.Y. Times, A-11 Jan. 11, 1985. AIDS has been characterized by the onset of severe opportunistic infections secondary to an effect on the body's immune system. Gottlieb, M. S. et al., Pneumocystis Carinic Pneumonia and Mucosal Candidiasis in previously healthy homosexual men: evidence of a new acquired cellular immuno-deficiency, N. Eng. J. Med. 305, 1426-1431 (1981). The disease has been found in male homosexuals, patients receiving blood products, intravenous drug addicts, and individuals originating from Haiti and Central Africa. Piot, P. et al. Acquired immunodeficiency syndrome in a heterosexual population in Zaire. Lancet 11, 65-69 (1984). The causative agent was suspected to be of viral origin as the epidemiological pattern of AIDS was consistent with a transmissable disease. At least three (3) retroviruses have been isolated from cultured T-cells of several patients with AIDS, or from white blood cells of persons at risk for the disease. A novel human retrovirus called lymphadenopathy-associated virus (LAV) was discovered and its properties were consistent with its etiological role in AIDS. That virus was isolated from a patient with lymphadenopathy and hence the name. Montagnier, L. et al. A New Human T-lymphotropic retrovirus: characterization and possible role in lymphadenopathy and acquired immune deficiency syndromes. In Human T-Cell Leukemia/Lymphoma Virus, R. C. Gallo, M. Essex and L. Gross, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory) pp. 363-370. Other human retroviruses, specifically two subgroups of the human T-cell leukemia/lymphoma/lymphotropic virus, types I and III have been isolated. (HTLV I: Poicsz, B. J. et al. PNAS (USA) 77, 7415 (1980)); (HTLV-III: Popovic, M. et al. Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 797-500 (1984)). Still another virus, the AIDS-associated retrovirus (ARV), was proposed as the causative agent. Levy, J. A. et al. Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225, 840-842 (1984)). Both the HTLV-III and ARV retroviruses display biological and sero-eidemiological properties similar to LAV. Levy et al., supra, Popovic et al. supra. As seen from the above, at least three (3) retroviruses have been postulated as the etiologic agent or AIDS: LAV: ARV; and, HTLV subtypes I and III.
LAV, HTLV III and ARV-II genomes have been molecularly cloned. Shaw, G. M. et al., Serological analysis of a subgroup of human T-lymphotropic retroviruses (HTLV III) associated with AIDS. Science 224, 503-505 (1984). Alizon, M. et al. Molecular Cloning of lymphadenopathy--associated virus. Nature, in press. The complete nucleotide sequence of the proviral genome of LAV, ARV and HTLV III has been determined. Ratner, L. et al. Complete nucleotide sequence of the AIDS virus, HTLV III. Nature 313, 277-284 (1985); Sanchez-Pescadov, R. et al. Nucleotide sequence and expression of an AIDS-associated retrovirus (ARV-2). Science 227, 484-492 (1985); and, Wain-Hobson, S. et al. Nucleotide sequence of the AIDS virus, LAV. Cell 40, 9-17 (1985).
One reason for the difficulty in determining the etiologic agent of AIDS was due to the reactivity of various retroviral antigens with serum samples from AIDS patients. For example, serum samples from AIDS patients have been shown to react with antigens of HTLV I and HTLV III. (HTLV-I: Essex, M., et al., "Antibodies to Cell Membrane Antigens Associated with Human T-Cell Leukemia Virus in Patients with AIDS", Science 220, 859(1983)); (HTLV-III: Sarngadharan, M. G. et al., "Antibodies Reactive With Human T-Lymphotropic Retroviruses (HTLV-III) in the Serum of Patients With AIDS", Science 224, 506-508 (1984)). Envelope gene products of HTLV demonstrated antigenicities cross-reactive with antibodies in sera from adult T-cell leukemia patients. Kiyokana, T. et al. Envelope proteins of human T-cell leukemia virus: Expression in Escherichia coli and its application to studies of env gene functions" PNAS (USA) 81, 6202-6206 (1984). Adult T-cell leukemias (ATL) differ from acquired immune deficiency syndrome (AIDS) in that HTLV-I causes T-cell malignancies, that is uncontrolled growth of T-cell. In AIDS rather than cell growth there is cell death. In fact this cytopathic characteristic of HTLV III was critical to determining ultimately the specific retroviral origin of the disease. Thus the etiologic agent of AIDS was isolated by use of immortalized human neoplastic T cell lines (HT) infected with the cytopathic retrovirus characteristic of AIDS, isolated from AIDS afflicted patients. Seroepidemiological assays using this virus showed a complete correlation between AIDS and the presence of antibodies to HTLV III antigens. Gallo et al. supra 1984; Sarngadharan et al. supra 1984; Schupbach et al. Serological Analysis of a subgroup of human T-lymphotropic retroviruses (HTLV III) associated with AIDS, Science 224, 503-505 (1984). In addition, nearly 85% of patients with lymphadenopathy syndrome and a significant proportion of asymptomatic homosexual men in AIDS endemic areas were also found to carry circulating antibodies to HTLV III. Taken together, all these data indicate HTLV III to be the etiologic agent for AIDS.
Until the successful culturing of AIDS virus using H-9 cell line the env AIDS protein of the AIDS virus had not been isolated, characterized or synthesized. This in major part is due to the fact that the virus is cytopathic and thus isolation of the virus was not possible. Popovic, M. et al., Detection, Isolation, and Continuous Production of Cytopathic Retroviruses (HTLV III) From Patients With AIDS and Pre AIDS, Science 224, 497-500 (1984). Once the human T-cell line resistant to the cytopathic effects of the virus was discovered, a molecular clone of proviral DNA could be achieved.
The need for a sensitive and rapid method for the diagnosis of AIDS in human blood and its prevention by vaccination is very great. Virtually all the assays/tests presently available are fraught with errors. In fact the Center for Disease Control (CDC) has indicated that presently available tests be used solely for screening units of blood for antibody to HTLV III. The CDC went further by stating that the presently available ELISA tests not be used for general screening of high risk populations or as a diagnostic test for AIDS. Federal Register 50(48), 9909, Mar. 12, 1985. The errors have been traced to the failure to use a specific antigenic protein of the etiologic agent for AIDS. The previously used proteins were derived from a viral lysate. Since the lysate is made from human cells infected with the virus, i.e. the cells used to grow the virus, the lysate will contain human proteins as well as viral proteins. Thus preparation of a pure antigen of viral protein is very difficult. The antigen used produced both false positive and false negative results. Budiansky, S., AIDS Screening, False Test Results Raise Doubts, Nature 312, 583(1984). The errors caused by the use of such lysate proteins/peptides can be avoided by using a composition for binding AIDS antibodies which is substantially free of the non-AIDS specific proteins. Compositions that are substantially pure AIDS envelope protein can be used as antigens. The AIDS envelope protein of the instant invention has been established to have conserved epitopes which permit its use to screen for, diagnose and/or prevent by vaccination the AIDS virus. The instant invention demonstrates that the envelope protein with its conserved epitopes includes all the variants which have been claimed as the sole etiologic agent.
The envelope AIDS protein of the present invention may be produced by conventionally known methods. The processes by which the novel protein may be produced can be divided into three groups: (1) chemical synthesis; (2) preparation of a gene prepared by chemical synthesis is inserted into a host and a protein is produced by the host; and (3) a gene obtained biotechnically is inserted into a host and a protein is produced by the host.
In one embodiment of this invention, recombinant DNA techniques are utilized by which env AIDS DNA from a natural source is introduced into a cell to produce the env AIDS protein. One method of obtaining DNA which encodes env AIDS is to read the genetic code in reverse and synthesize an oligodioxynucleotide which should encode the env AIDS amino acid sequence. As the env protein has not been isolated or characterized this approach cannot be pursued.
Alternatively gene expression can be obtained using recombinant DNA technology if DNA isolated from natural sources is used instead of synthetic DNA.